Phosphate linked cytosine-guanine (CpG) dinucleotides are statistically underrepresented in the genomes of higher eukaryotes, including mammals. The dinucleotide is reportedly found at only 5-10% of its predicted frequency. The majority of CpG dinucleotides that do remain in the human genome are normally located within repetitive sequences that are characterized by low gene expression levels and exhibit methylation at the cytosine residues.
CpG islands, on the other hand, represent genomic sequences that contain clusters of CpG dinucleotide. CpG islands may be associated with the promoter region or 5′ end of coding sequences or may be present within introns or in genomic regions that are not known to be associated with coding sequences. They may be unmethylated or methylated in normal tissues and the methylation pattern may be used to control tissue specific expression and the expression of imprinted genes. Methylation of CpG islands within promoter regions can result in the downregulation or silencing of the associated gene. An increase in methylation of normally unmethylated islands is observed in aging tissues even as the overall methylcytosine content of the DNA is reduced. The aberrant methylation pattern is more pronounced in cancer cells with increased methylation or hypermethylation detected in various cancer tissues. CpG islands may be methylated to varying densities within the same tissue. Thus, aberrant methylation of cytosines within CpG islands can be a primary epigenetic event that acts to suppress the expression of genes involved in critical cellular processes, such as DNA damage repair, hormone response, cell-cycle control, and tumor-cell adhesion/metastasis, leading to tumor initiation, progression and metastasis (Li et al., Biochim. Biophys. Acta, 1704: 87-102 (2004)). It has been proposed that a unique profile of promoter hypermethylation exists for each human cancer in which some gene changes are shared and other gene changes are cancer-type specific (Esteller et al., Cancer Res., 61: 3225-3229 (2001)). Given that aberrant methylation represents new information not normally present in genomic DNA and that aberrant methylation is a common DNA modification and affects a large number of genomic targets, it is feasible to develop diagnostic and prognostic tests based on information obtained from multiple target CpGs. Such tests may be based on CpGs that are aberrantly hypermethylated or hypomethylated in the diseased tissues. They may also be based on changes in methylation density in CpG islands as long as the changes corrolate with the presence of cancer.
Prostate cancer, for example, which is the most common malignancy and the second leading cause of death among men in the U.S. (Li et al. (2004), supra), has been found to be associated with the methylation of CpG islands in the promoters of over 30 genes, in particular the CpG island of the glutathione S-transferase P1 (GSTP1) gene. GSTP1 methylation has been detected in over 50% of DNA recovered from urine and plasma of prostate cancer patients (Goessl et al., Ann. N.Y. Acad. Sci., 945: 51-58 (2001); Cairns et al., Clin. Cancer Res., 7: 2727-2730 (2001); Jeronimo et al., Urology, 60: 1131-1135 (2002); and Gonzalgo et al., Clin. Cancer Res., 9: 2673-2677 (2003)). However, if diagnosis of prostate cancer relied solely on the detection of the methylation of the CpG island in the GSTP1 gene, the theoretical limit of the sensitivity of such a test would only be approximately 90%. GSTP1 is also methylated in prostatic intraepithelial lesions (PIN) which may lead to a false positive diagnosis. Some CpG islands are methylated in prostate cancer and other diseases of the prostate, such as benign prostatic hyperplasia (BPH). They may even exhibit some degree of methylation in normal aging prostates. Such markers may not be suitable individually for prostate cancer diagnosis. Therefore, a panel of markers is required to achieve the sensitivity and specificity needed for a clinical test.
The prostate-specific antigen or PSA test continues to be widely used in the early detection of prostate cancer. While the PSA test has resulted in the majority of prostate cancer cases being diagnosed in asymptomatic men (Mettlin et al., Cancer, 83(8): 1679-1684 (1998a); Mettlin et al., Cancer, 82(2): 249-251 (1998b); Humphrey et al., J. Urol., 155: 816-820 (1996); and Grossfeld et al., Epidemiol. Rev., 23(1): 173-180 (2001)), the PSA test suffers from poor specificity, which can be as low as 33% when a PSA cut-off level of 2.6 ng/ml is used (Thompson et al., N. Engl. J. Med., 350: 2239-2246 (2004)), even though the sensitivity can be as high as 83%. The poor specificity of the PSA test is a direct result of increased secretion of PSA in other diseases of the prostate, such as BPH and prostatitis. Thus, an elevated PSA level indicates the need for additional screening in the form of needle biopsy. Ultimately, the results of needle biopsies lead to the diagnoses of prostate cancer.
Over 1 million needle biopsies of prostates are performed each year at a cost of about $1,500 each and much discomfort to the patient. However, less than 200,000 of these result in a diagnosis of prostate cancer. Therefore, the majority of needle biopsies are being performed needlessly.
In view of the above, there is a need for non-invasive methods of diagnosing and prognosticating cancer, such as prostate cancer, that reduce the cost and suffering associated with currently available cancer screening methods. It is an object of the invention to provide materials and methods for non-invasive diagnosis and prognosis of cancer, such as prostate cancer. This and other objects and advantages, as well as additional inventive features, will become apparent from the detailed description provided herein.